Assays and probes with enzyme labels

ABSTRACT

Probes comprise S1 and P1 nuclease (as an enzyme label) linked to a specific binding member such as a nucleotide sequence or an antibody. Such probes are useful for sandwich assays. As compared with known probes using alkaline phosphatase as a label, advantages include relative insensitivity to phosphate and elevated temperature and reduced risk of nonspecific binding.

TECHNICAL FIELD

[0001] This invention relates to probes comprising enzyme labels andspecific binding members such as antibodies and single-stranded nucleicacids, and assays employing such probes.

BACKGROUND ART

[0002] The use of enzymes as labels in a wide variety of clinical,veterinary and environmental diagnostic assays including enzymeimmunoassays and nucleic acid probe-based assays is well known. Oneexample of the use of these employs a sandwich format in which animmobilized antibody, antigen or nucleic acid is used to recognize andbind to a portion of the molecule to be detected. An appropriateenzyme-labelled antibody or nucleic acid probe is then introduced whichbinds to a different portion of the complex to be measured. This resultsin the formation of a complex immobilized to the solid surface which islabelled with the enzyme. After several washing steps to remove alltraces of the original sample and the excess unbound labelled moiety, asubstrate for the enzyme is introduced, and the presence of the enzymedetected by its action on a substrate to produce a change in colour,fluorescence, redox state, or to produce light.

[0003] It is important that enzymes employed as labels catalyze areaction which has an easily detectable product, and have a highturnover number to allow sensitive detection: horseradish peroxidase andalkaline phosphatase are most common. Although sensitivechemiluminometric assays for horseradish peroxidase have been describedwhich allow small amounts of enzyme to be detected, problems associatedwith its use include lack of enzyme and substrate stability and thepresence of endogenous peroxidases in samples.

[0004] For alkaline phosphatase, enzyme amplification cycles have beendescribed which further reduce the amount of enzyme which can bedetected, thereby extending the detection limit. For example, in U.S.Pat. No. 5,445,942 to Rabin et al., entitled “Amplification Assay forhydrolase enzymes”, a method is described for detecting a hydrolaseenzyme able to hydrolyze a synthetic derivative of FAD substituted insuch a way that it yields FAD when hydrolyzed. The FAD produced forms anactive holoenzyme from the corresponding apoenzyme. This approach allowsthe detection of small amounts of alkaline phosphatase in short periodsof time. For example, we have used such an amplification system in whichthe apoenzyme is apo-D-amino acid oxidase to measure 0.1 amol ofalkaline phosphatase in less than 30 minutes (Harbron S, et al., Anal.Biochem. (1992) 206: 119-124).

[0005] However, the use of alkaline phosphatase as the label enzyme hasa number of shortcomings: its large size (MW=140,000) means that it cansterically hamper the association of the antibody or nucleic acid probewith its target; its nature as a membrane-associated protein means thatit binds non-specifically to many surfaces; it is very sensitive to thepresence of phosphate carried over from previous assay stages; it haslimited stability at the temperatures often used in nucleic acidhybridization steps; and it is a commonly occurring enzyme in manytissues and occurs in the environment at large as a component ofbacteria and other microorganisms. Rabin et al. describe the use of theamplification assay for the detection of sulphatases, carboxylesterases,acetylesterase and venom phosphodiesterase which may obviate some ofthese problems, but they do not teach that the approach could be usedfor the assay of enzymes of the nuclease class, such as nuclease S1 andnuclease P1. It is known that nuclease P1 hydrolyses Coenzyme A(Fujimoto et al., Agr. Biol. Chem. (1974) 38: 1555-1561).

[0006] EP-A-401,001 concerns novel dioxetanes having a substituent-X-Y-Z where Z and Y are protecting groups which are removablesuccessively, leading to chemiluminescence. For a sandwich assay, Z maybe removed by a first triggering enzyme E1 which is directly orindirectly bound to an antigen, antibody or nucleic acid probe. E1 maybe a nuclease.

[0007] Further examples of assays involving enzyme-containing probes areprovided by EP-A-0,304,934, U.S. Pat. No. 5,563,063, WO-A-96/41015,WO-A-90/00252, EP-A-0,061,071, EP-A-0,124,124, EP-A-0,516,948 andGB-A-2018986.

DISCLOSURE OF INVENTION

[0008] We have discovered that both nuclease S1 and nuclease P1 canhydrolyze the synthetic analogue of FAD in which the 3′ hydroxyl groupon the ribose moiety of FAD is esterified with phosphoric acid to give3′FADP, thereby giving a new means of assaying these enzymes in anextremely rapid and sensitive fashion. But Fujimoto et al. also showedthat nuclease P1 hydrolyses single stranded DNA and RNA, which wouldindicate that this enzyme is unsuitable for labelling nucleic acidprobes. In fact, the prior art teaches that nucleases are used fordegrading nucleic acids: thus U.S. Pat. No. 5,145,780, to Oishi and Aoidescribes an enzyme preparation produced by a fungus such asTrichoderma, Aspergillus and Fusarium which contains a nuclease that isactive even after heating at 100° C. for 30 minutes. This enzymepreparation may be effectively used when it is necessary to decomposenucleic acids at elevated temperature over a prolonged period. U.S. Pat.No. 5,006,472, to Dove and Mitra, discloses a method for purifying rDNAor monoclonal antibody culture products by using nuclease enzymetreatment to degrade undesirable residual nucleic acids to a molecularsize or charge range sufficiently different from the product to bepurified so that this difference can be exploited in a subsequentpurification step (e.g. precipitation, size exclusion chromatography orion exchange chromatography).

[0009] Although it would not therefore be expected that nuclease P1 andnuclease S1 could be used to label nucleic acids, Fujimoto et al.demonstrated that the ability of nuclease P1 to hydrolyzesingle-stranded nucleic acids was pH dependent, and we have found thatpH values greater than 7.0 allow the labelling of nucleic acids withthese nucleases.

[0010] Broadly, the present invention relates to the use of P1 and S1nucleases as enzyme labels for assays. Thus in one aspect the inventionprovides a probe which comprises a nuclease (particularly P1 or S1)coupled to a specific binding member (“sbm”) (generally an antibody or asingle-stranded nucleic acid). The nuclease is preferably covalentlyattached to the sbm.

[0011] In another aspect the invention provides a method of producing aprobe which comprises coupling a nuclease to an sbm.

[0012] In further aspects the invention provides an assay methodemploying a probe according to the first aspect, and a kit for carryingout such an assay.

[0013] A preferred type of sbm is antibodies (particular IgG antibodies)and functional fragments thereof capable of binding to a target in anassay procedure.

[0014] Another preferred type of sbm is nucleic acids (DNA, RNA oranalogues thereof), generally oligonucleotides. The nucleic acid may beproduced with a derivatised 5′-end (e.g trityl-hexyl thiol derivatised)to facilitate coupling to a nuclease which has been rendered susceptibleto disulphide exchange, e.g. being 2-pyridyl disulphide activated.

[0015] Preferred embodiments of the invention may enable one to achieveone or more of the following objects and advantages:

[0016] (a) to provide an enzyme label which is small and which does notinterfere with the association of antibody and antigen, nor ofcomplementary strands of nucleic acid;

[0017] (b) to provide an enzyme label which may be easily conjugated toantibodies and nucleic acids using well-known methodologies;

[0018] (c) to provide an enzyme label which is not membrane associatedin its natural state, and which is secreted into the growth medium, andwhich therefore has a low level of non-specific binding to solidsurfaces;

[0019] (d) to provide an enzyme label which is largely insensitive tothe presence of phosphate, allowing it to be used in automated assaymachinery in which phosphate-containing washing solutions are routinelyused;

[0020] (e) to provide an enzyme label which has good temperaturestability, allowing it to be used at high temperature, particularly innucleic acid assays;

[0021] (f) to provide an enzyme label which is not a commonly occurringenzyme, thereby avoiding contamination from endogenous enzyme in thesample.

[0022] Further objects and advantages are to provide the use as enzymelabels of enzymes which are commercially available, which are notinhibited by phosphate monoesters which may be included in the assaysolution to prevent endogenously occurring phosphatases from hydrolysing3′FADP, which do not hydrolyze single-stranded nucleic acids at the pHemployed in the assay solution, and which can be assayed using an enzymeamplification system.

[0023] Some embodiments of the invention will be described in moredetail, by way of example, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

[0024]FIG. 1 slows a standard curve for the 3′FADP-based enzymeamplification assay of nuclease P1 and S1;

[0025]FIG. 2 is a graphic comparison of the effect of phosphate on the3′FADP-based enzyme amplification assay of alkaline phosphatase andnuclease P1;

[0026]FIG. 3 is a graphic comparison of the effect of p-nitrophenylphosphate on the activity of nuclease P1 and endogenous phosphatase(“FADPase”) activity measured using the 3′FADP-based enzymeamplification assay;

[0027]FIG. 4 is a graphic comparison of a nuclease P1-based enzymeimmunoassay for measles (using the 3′FADP-based enzyme amplificationassay and measuring the absorbance at 520 nm) with an alkalinephosphatase-based enzyme immunoassay, (using p-nitrophenyl-phosphate andmeasuring the absorbance at 405 nm); and

[0028]FIG. 5 is a graph showing the results of a microtitre plate-basedgene probe assay using a nuclease P1-labelled probe. The absorbance wasmeasured after 20 minutes incubation with the 3′FADP-based enzymeamplification assay.

MODES FOR CARRYING OUT THE INVENTION

[0029] One preferred type of embodiment of the present invention employsthe enzyme nuclease P1 covalently attached to an antibody. The covalentattachment may be achieved by a number of well-known methods using awide range of heterobifunctional reagents. For example, the method ofCarlsson et al. (Biochem J (1978) 173: 723-737) may be used: nuclease P1is reacted with 3-[(2)-pyridyldithio]propionic acid N-hydroxysuccinimideester (SPDP) to give a 2-pyridyl disulphide-activated enzyme. This ismixed with an IgG antibody, and a disulphide exchange reaction yields anuclease P1-IgG antibody conjugate.

[0030] This conjugate may, for example, be used in a sandwichimmunoassay in which an antibody immobilized on a microtitre plate bindsa target antigen from a sample, and the nuclease P1-IgG antibodyconjugate binds to another site on the antigen, producing an immobilizedcomplex labelled with nuclease P1. Following a number of washing stepsnuclease P1 which has become immobilized in this way can be detectedusing the prosthetogenic amplification system of Rabin et al. For thisassay, a solution containing buffer, 3′FADP, apoglucose oxidase,glucose, horseradish peroxidase and its substrates are added. 3′FADP ishydrolyzed by nuclease P1 to yield FAD which is bound by apoglucoseoxidase. The hologlucose oxidase thus formed oxidizes glucose to producehydrogen peroxide, which is in turn a substrate for horseradishperoxidase, yielding a coloured product conveniently quantitated in amicroplate reader. To eliminate signal caused by endogenous phosphataseremaining after the washing step, which would also hydrolyze 3′FADP togive FAD, a phosphatase substrate such as p-nitrophenyl phosphate or2-glycerophosphate, may be added. The phosphatase contaminant willhydrolyze this in preference to 3′FADP.

[0031] Another preferred type of embodiment of the present inventionemploys the enzyme nuclease P1 covalently attached to a nucleic acid.The nucleic acid may be DNA or RNA or an analogue thereof. The nucleicacid may be an oligonucleotide produced by solid-phase chemistry by aNucleic Acid synthesizer having a trityl-hexyl thiol derivatized 5′-end.This allows disulphide exchange with the 2-pyridyl disulphide-activatedenzyme described above to yield a nuclease P1-oligonucleotide conjugate.

[0032] This conjugate may, for example, be used in a sandwichhybridization assay in which an oligonucleotide immobilized on amicrotitre plate binds a single-stranded target nucleic acid from asample denatured with alkali. After annealing and neutralisation of thealkali, the nucleate P1-oligonucleotide conjugate binds to another siteon the target nucleic acid, producing an immobilized complex labelledwith nuclease P1. Following a number of washing steps nuclease P1 whichhas become immobilized in this way can be detected using theprosthetogenic amplification system of Rabin et al. For this assay, asolution containing buffer, 3′FADP, apoglucose oxidase, glucose,horseradish peroxidase and its substrates are added. 3′FADP ishydrolyzed by nuclease P1 to yield FAD which is bound by apoglucoseoxidase. The hologlucose oxidase thus formed oxidizes glucose to producehydrogen peroxide, which is in turn a substrate for horseradishperoxidase, yielding a coloured product conveniently quantitated in amicroplate reader. To eliminate signal caused by endogenous phosphataseremaining after the washing step, which would also hydrolyze 3′FADP togive FAD, a phosphatase substrate such as p-nitrophenyl phosphate or2-glycerophosphate, may be added. The phosphatase contaminant willhydrolyze this in preference to 3′FADP.

EXAMPLE 1 Standardization of Nuclease P1

[0033] Nuclease P1 (1 mg; obtained from Sigma Chemical Company, batchno: 107F0799) was dissolved in 1 ml of water to give a concentration of22.7 μM and stored at 4° C. The activity of this solution was assayed inthe following mixture: 0.16 mM NADH, 1 mM ATP, 1 mM PEP, 1 mM MgSO₄, 20mM KCl, 0.5 mM adenosine 3′,5′-bisphosphate, 1 U pyruvate kinase, 1 Ulactate dehydrogenase and 1 U myokinase in 50 mM HEPES buffer, pH 7.2,in a total volume of 1 ml. From the change in absorbance at 340 nm theactivity of nuclease P1 was solution was found to be 320 U/ml, assuminga molar extinction coefficient of 6220 for NADH.

EXAMPLE 2 Amplification Assay of Nuclease P1 and Nuclease S1

[0034] A solution of nuclease P1 standardized according to Example 1 wasserially diluted in 50 mM citrate buffer adjusted to pH 6.5 with NaOH.The assay mixture contained 20 μM 3′FADP, 0.1 mM 4-aminoantipyrine, 2 mMDHSA, 1 μg horseradish peroxidase, 0.1 M glucose and 0.1 μM apoglucoseoxidase in a total volume of 0.1 ml. The change in absorbance wasmonitored at 520 nm in a Dynatech MR7000 plate reader fitted with athermostatically controlled plate holder set to 25° C. FIG. 1 shows theperformance of the nuclease P1 assay. After a 15 minute assay period,the detection limit (defined as 3 times the standard deviation of thebackground reading) was 0.2 amol. Nuclease S1 was assayed in a similarmanner, and the detection limit was 4 amol (FIG. 1).

EXAMPLE 3 Effect of Phosphate on the Activity of Nuclease P1 andAlkaline Phosphatase

[0035] Phosphate buffer, pH 6.5, was added to the reaction mixturedescribed in Example 2 to give a final concentration of phosphateranging from 0 to 10 mM. The effect of phosphate on the activity ofnuclease P1 is shown in FIG. 2. This was compared with the effect ofphosphate on alkaline phosphatase. The same assay mixture was used, but0.1 M Tris buffer, adjusted to pH 8.9 with HCl was used instead of thecitrate buffer, and the phosphate buffer which was added was alsoadjusted to pH 8.9. Clearly, phosphate has less of an effect on nucleaseP1 than on alkaline phosphatase.

EXAMPLE 4 Effect of p-Nitrophenyl Phosphate on the Activity of NucleaseP1

[0036] The effect of p-nitrophenyl phosphate on the activity of nucleaseP1 was investigated by adding p-nitrophenyl phosphate to the assaymixture described in Example 2, to give a final concentration rangingbetween 0 and 10 mM. The effect of the added p-nitrophenyl phosphate onthe background color generation in the absence of nuclease P1 was alsonoted. This background is due to endogenous “FADPase” in the apoglucoseoxidase used in the assay. At concentrations up to approximately 5 mM,added p-nitrophenyl phosphate has no effect on nuclease P1, but reducesthe background signal by 80-100% (FIG. 3).

EXAMPLE 5 Oligonucleotide Synthesis

[0037] Oligonucleotide were synthesized on a Cyclone™ DNA synthesizerusing the Expedite™ chemistry. The DNA to be immobilized on a microtitreplate, known as the capture DNA probe, was designed to capture a plasmidcontaining the 5′-end of the gene encoding human pancreaticribonuclease, including the bovine leader sequence (see Taylorson et al.WO96/2001). This plasmid also had R4A, K6E and K66E mutations. Thesequence was:

[0038] 5′-GAATTCCCATGGCGAAGGAATCCGCTGCCGCTAAA-3′

[0039] The DNA to be labelled with nuclease P1, known as the reporterprobe, was complimentary to a region in the middle of the ribonucleasegene containing the K66E mutation. This probe was derivatized at the 5′end with a trityl-hexyl thiol group to facilitate linkage to nucleaseP1. The sequence was:

[0040] 5′-GGTCACCTGCGAAAACGGGCAGG-3′

[0041] The oligonucleotide were freeze-dried and stored at 4° C. untilrequired.

EXAMPLE 6 Derivatization of Nuclease P1

[0042] Nuclease P1 (5 mg) was dissolved in 0.5 ml 0.1 M sodiumbicarbonate pH 7.5 containing 0.1 M sodium chloride and desalted by gelfiltration on Sephadex G25 (NAP-5 column, Pharmacia) equilibrated withthe same buffer. This enzyme solution was incubated with a 50-fold molarexcess of 3-(2)-pyridyldithio)-propionic acid N-hydroxysuccinimide ester(SPDP) at room temperature for 30 minutes. Unreacted SPDP was removed bygel filtration on Sephadex G25 (NAP 10 column, Pharmacia) equilibratedwith the bicarbonate buffer. The 2-pyridyl disulphide-activated nucleaseP1 was stored at 4° C.

EXAMPLE 7 Conjugation of Nuclease P1 to Antihuman IgG

[0043] The 2-pyridyl disulphide-activated nuclease P1 prepared accordingto Example 6 was transferred to 0.1 M sodium acetate buffer, pH 4.5,containing 0.1 M sodium chloride by gel filtration on Sephadex G 25.Antihuman IgG (γ-chain specific) was dissolved in the acetate buffer togive a concentration of 3 mg/ml, and desalted by gel filtration onSephadex G 25 (NAP 5 column, Pharmacia) equilibrated with the samebuffer. Activated nuclease P1 was mixed with the IgG solution at a molarratio of 3:1, and incubated at room temperature for 45 minutes, and thenat 4° C. for a further 16 hours. The conjugate was transferred to 20 mMbis-Tris buffer, pH 6.5, containing 1 mM CHAPS by chromatography onSephadex G25 equilibrated with the same buffer, prior to purification byion exchange chromatography on a Pharmacia Mono-Q column. The conjugatewas eluted in the same buffer containing 20 mM sodium chloride.

EXAMPLE 8 Conjugation of Nuclease P1 to an Oligonucleotide

[0044] Nuclease P1 was linked to 2-pyridyl disulphide as described inExample 6 and stored in 0.1 M sodium bicarbonate, pH 7.5, containing 0.1M sodium chloride at 4° C. The reporter oligonucleotide of Example 5 wasdissolved in 0.5 ml 0.1 M sodium bicarbonate buffer, pH 7.5, containing0.1 M sodium chloride to give a final concentration of 0.36 mM. This wasincubated with activated nuclease P1 prepared according to Example 6 ata mole ratio of 1:2 at room temperature for 45 minutes, followed by anincubation at 4° C. for 16 h.

[0045] The conjugate was transferred to 20 mM bis-Tris propane buffer,pH 7.5, containing 1 mM CHAPS by chromatography on Sephadex G25, andpurified by ion-exchange chromatography on a Pharmacia Mono Q column. Asodium chloride gradient in the same buffer was used applied to thecolumn and the conjugate was eluted at a molar concentration of 0.25 M.

EXAMPLE 9 Enzyme Immunoassay Employing Nuclease P1-Conjugated AntihumanIgG

[0046] Standard solutions containing human serum IgG antibodies tomeasles were incubated in microtitre plates coated with purified measlesantigen (Edmonston strain, obtained from Sigma Chemical Co as the SIAmeasles IgG assay kit) for 30 min at room temperature. Each well waswashed 5 times with a buffered solution containing surfactant (assupplied in the kit from Sigma). 200 μl of a 2 nM solution of nucleaseP1 conjugate in 20 mM bis-Tris buffer, pH 6.5, containing 1 mM CHAPS wasadded to each well. The plate was covered and incubated at roomtemperature for 30 minutes. Each well was washed 5 times with thebuffered solution containing surfactant to remove excess conjugate. Thebound conjugate was quantitated using the amplification assay of Example2, with an assay time of 5 minutes.

[0047]FIG. 4 compares the absorbance produced using the amplificationassay for nuclease P1 described in Example 2 with that obtained using analkaline phosphatase-labeled antibody assayed using p-nitrophenylphosphate (the method normally used with the SIA measles IgG assay kit).The nuclease P1-based enzyme immunoassay is approximately 5 times moresensitive than the alkaline phosphatase/p-nitrophenyl phosphatase basedsystem of the kit.

EXAMPLE 10 Covalent Attachment of Capture Oligonucleotide to aMicrotitre Plate

[0048] The capture oligonucleotide of Example 5 was immobilized to thewalls of a Covalink NH microtitre plate (Nunc) as described by Rasmussenet al. (Anal Biochem (1991) 198: 138-142). The capture oligonucleotidewas dissolved in 1 ml sterile water and its concentration determinedfrom its absorbance at 260 nm. It was phosphorylated using T4polynucleotide kinase in the presence of a 5-fold molar excess of ATP at37° C. for 30 minutes. The reaction was terminated by heating to 95° C.followed by rapid cooling on ice. The solution of oligonucleotide wasdiluted with 143 mM 1-methylimidazole, pH 7.0, to give a finalconcentration of 1.3 μM, and 70 μl of this solution, containing 91 fmolof oligonucleotide, was added to each well of the Covalink NH microtitreplate. This was followed by 30 μl of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the plate was sealedand incubated at 50° C. for 5 hours. Reaction solution was then removedfrom the wells and the plate was washed 3 times with 0.4 M sodiumhydroxide containing 0.25% (w/v) SDS at 50° C., followed by a further 3washes at room temperature with 10 mM Tris-HCl buffer, pH 8.0,containing 1 mM EDTA. The plates were stored at 4° C. until required.

EXAMPLE 11 Hybridization and Detection of Plasmid DNA

[0049] 50 pg of λDNA, dissolved in 95 μl sterile water was added to eachwell of the microtitre plate prepared in Example 10. This served as acontrol for non-complementary binding. A further 5 μl of a known amountof the plasmid containing the human RNase mutant and 10 μl 1 M sodiumhydroxide were added. This mixture was incubated at room temperature for10 minutes to denature the plasmid before neutralisation with 8 μl of0.5 M sodium citrate buffer, pH 3.0, containing 2.21 M sodium chlorideand 0.1% Tween 20.

[0050] 50 μl (34 fmol) of the nuclease P1-conjugated reporter probe,prepared according to Example 8, dissolved in 0.1 M Tris-HCl buffer, pH7.5, containing 7 mM zinc sulphate, 1% (w/v) PVP, 0.1%N-lauroylsarkosine and 150 mM sodium chloride, was added to each well.After hybridization at 40° C. for 1 hour, the wells were washed 6 timeswith 20 mM Tris-HCl buffer, pH 7.5, containing 7 mM zinc sulphate, 1%(w/v) PVP, 0.1% N-lauroylsarkosine and 150 mM sodium chloride.

[0051] The amount of conjugate hybridized to the microtitre plate wasquantitated using the amplification assay described in Example 2. FIG. 5shows that as little as 35 amol of DNA can be detected in this way in atotal assay time of 90 minutes (10 minutes denaturation, 60 minuteshybridization and 20 minutes of amplification assay).

[0052] It will be seen that the enzyme labels used in preferredembodiments of the present invention are smaller, more stable, lessprone to non-specific binding, and give less background from endogenousphosphatases than those previously described.

[0053] The above description contains many specificities which shouldnot be construed as limitations on the scope of the invention, butrather as exemplifications of preferred embodiments thereof. Many othervariations are possible. For example, apo-D-amino oxidase could be usedinstead of apo-glucose oxidaze, as the apoenzyme.

1. A probe for use in an assay comprising a nuclease selected from P1and S1 nucleases coupled to a specific binding member (“sbm”).
 2. Aprobe according to claim 1 wherein the sbm is an antibody or afunctional fragment thereof.
 3. A probe according to claim 1 wherein thesbm is an IgG antibody or a functional fragment thereof.
 4. A probeaccording to claim 1 wherein the sbm is a single-stranded nucleic acid.5. A probe according to any of claims 1 to 4 wherein the nuclease iscovalently attached, directly or indirectly, to the sbm.
 6. A method ofproducing a probe according to any preceding claim which comprisescoupling a nuclease to an sbm.
 7. A method according to claim 6 whereinthe coupling is between a nucleic acid with a derivatised 5′-end and anuclease which has been rendered susceptible to disulphide exchange. 8.An assay employing a probe according to any of claims 1 to 5 comprisinga nuclease and a first sbm, wherein i) a sample believed to contain ananalyte is brought into contact with a carrier having a second sbmimmobilised to it so that analyte in the sample binds to the second sbmand is thus bound to the carrier, said first and second sbm's beingselected such that they bind to different sites on the analyte; ii) thebound analyte is contacted with the probe so that the probe binds to theanalyte via the first sbm; and iii) the bound probe is contacted with asignal system such that the activity of the nuclease of the bound probeleads to a detectable signal.
 9. An assay according to claim 8 in whichstep (iii) is carried out in the presence of phosphate.
 10. An assayaccording to claim 9 wherein said phosphate comprises inorganicphosphate.
 11. An assay according to claim 9 or claim 10 wherein saidphosphate comprises a phosphate monoester.
 12. An assay according to anyof claims 8 to 11 wherein the probe comprises antihuman IgG antibody (asthe first sbm) the second sbm is measles antigen, and the analyte ishuman serum IgG antibodies to measles.
 13. An assay according to any ofclaims 8 to 11 wherein the probe comprises an oligonucleotide and theanalyte is single-stranded dna.
 14. An assay according to any of claims8 to 13 wherein said signal system comprises an amplification system.15. An assay according to claim 14 wherein said amplification systemcomprises an apoenzyme which is convertable into a holoenzyme byinteraction with an accessory subunit; and a masked form of said subunitwhich is convertible into its active unmasked form by the action of thenuclease of the probe.
 16. An assay according to claim 15 wherein saidsubunit is FAD and said masked form is 3′-FADP.
 17. An assay accordingto claim 15 or 16 wherein said apoenzyme is apo-glucose oxidase orapo-D-amino oxidase.